To determine the relationship between GPN-sensitive and Ins(1,4,5)P
3-sensitive stores, several experiments were initiated. In the first series, an attempt was made to examine the extent of the GPN response before and after emptying Ins(1,4,5)P
3-sensitive stores. In previous studies, in the absence of extracellular Ca
2+, UTP has been shown to deplete Ins(1,4,5)P
3-sensitive Ca
2+ stores such that the [Ca
2+]
i increase was diminished in response to subsequent exposure to cyclopiazonic acid (10 μM; ER Ca
2+-ATPase inhibitor) or to a second exposure to UTP itself.
27 In the absence of extracellular Ca
2+, initial exposure to UTP (100 μM) mobilized [Ca
2+]
i (Δ[ratio]
Peak = 0.62 ± 0.15;
n = 8), but subsequent exposure to GPN (200 μM) did not result in a detectable increase in [Ca
2+]
i (data not shown). By contrast, prior exposure to GPN did not prevent UTP-induced [Ca
2+]
i mobilization (
n = 10; data not shown). In the presence of extracellular Ca
2+, however, responses to GPN after exposure to UTP were significant (Δ[ratio]
peak = 0.27 ± 0.12;
n = 4), but were smaller compared with the response obtained when cells were previously exposed to GPN (
P < 0.01). These data show that GPN-sensitive stores are sensitive to UTP. To determine whether this sensitivity is associated with Ins(1,4,5)P
3-sensitive stores, the latter were depleted by thapsigargin, an irreversible ER Ca
2+-ATPase inhibitor. As shown in
Figure 9 (top), GPN induced a [Ca
2+]
i increase in the continued presence of thapsigargin. The extent of Ca
2+ increase was close to that found in the absence of thapsigargin (summarized in
Fig. 9 , bottom), indicating that the GPN response was independent of Ins(1,4,5)P
3. To determine whether GPN caused an increase of Ins(1,4,5)P
3, its potential release through the breakdown of membrane phospholipids was blocked by the inhibition of phospholipase C (PLC) with U73122. The time of exposure required to completely inhibit PLC-mediated Ca
2+ release with U73122 was first determined using UTP to stimulate release from Ins(1,4,5)P
3-sensitive stores. An exposure time of 7 minutes or more completely blocked the PLC-mediated increase in Ca
2+ by UTP (100 μM;
Fig. 10C ).
Figure 10D summarizes similar experiments conducted for different durations. Based on these findings, cells were pretreated with U73122 (10 μM) for 7 to 9 minutes and then exposed to GPN in the continued presence of the inhibitor. A typical response to GPN in the presence of U73122 is shown in
Figure 11A , and the results of all similar experiments are summarized in
Figure 11B . Although the extent of the GPN response was reduced significantly (
P < 0.01) compared with the control
(Fig. 11B) , the GPN response was still much larger than that to UTP, which at 100 μM did not produce any detectable increase in Ca
2+ after pretreatment with U73122
(Figs. 10C 10D) . A significant decrease in the GPN response compared with the control appeared to be nonspecific, because pretreatment with U73343 (10 μM), an inactive analogue of U73122, also caused similar decreases in response to UTP and GPN
(Figs. 12) .